PCR Flashcards
(90 cards)
1
Q
• Started in 1990 involving about
30 different institutions
A
THE HUMAN GENOME PROJECT
2
Q
HUMAN GENOME PROJECT GOAL
A
• Goal: Fully sequence the entire human genome
3
Q
THGP
• Initial completion:
• Mission Accomplished:
A
April 2003
MAY 2021
4
Q
HUMAN GENOME
Toal size
A
~ 3.2 billion bp
5
Q
Studying Specific Sequences
•_______ - the genome is LARGE
•_______ - amount of DNA
samples are VERY small
A
SPECIFICITY
AMPLIFICATION
6
Q
Came upon a way to double his test target DNA by giving him 2^1 copies
Repeating N times would yield: 2^N
By adding an oligonucleotide primer with the 4 dNTPs and DNA polymerase in a chain reaction
A
Dr. Kary Mullis
7
Q
Dr. Kary Mullis
______Nobel Prize Winner in
Chemistry for the_______
A
1993
Polymerase Chain Reaction
8
Q
PCR STEPS (3)
A
1 Denaturation
2 Annealing
3 Extension
9
Q
• Double-stranded DNA is separated into 2 single strands
A
Denaturation
10
Q
DENATURATION
• temperature:
• Time:
• Larger template = longer time
A
94-96°C
several seconds to minutes
11
Q
• MOST CRITICAL step for specificity
A
Annealing
12
Q
Primers hybridize to their complementary DNA sequences
A
ANNEALING
13
Q
ANNEALING
• Temperature:
• Starting point is determined using the_______
A
50-70°C
Tm of the primer sequences
14
Q
Melting Temperature (Tm)
Formula 1 and 2
A
Tm = 81.5 deg C + 16.6 log M + 0.41(%GC) - 0.61% formamide) - (600/n)
Tm = 81.5 deg C + 0.41(%GC) - (675/n)
15
Q
• Performed by DNA polymerase
• Polymerase synthesizes a copy of the template DNA
• Catalyzes formation of the phosphodiester bonds between dNTPs and the 3’ end of the primer
A
EXTENSION
16
Q
EXTENSION
• Performed by______
• Polymerase synthesizes a copy of the______
• Catalyzes formation of the _______between dNTPs and the 3’ end of the primer
A
DNA polymerase
template DNA
phosphodiester bonds
17
Q
EXTENSION
• Temperature:_______
• End result:______
A
68-72°C
2x template number
18
Q
Temperature (°C)
Time (sec)
Denaturation
A
90-96
20-60
19
Q
Temp
Time
Annealing
A
50-70
20-90
20
Q
Temp
Time
Extension
A
68-75
10-60
21
Q
• Single-stranded DNA fragments
A
PRIMERS
22
Q
PRIMERS
• BP LENGTH?
A
20-30 bp long
23
Q
• Works like the primers in vivo
• Determine the specificity of PCR
• Contain sequences complementary to sites flanking the region of interest
A
PRIMERS
24
Q
• 5’ to the sequences amplified
• Hybridizes with the minus strand
A
• FORWARD PRIMER
25
• 3' to the sequences amplified
• Hybridizes with the plus strand
• REVERSE PRIMER
26
• FORWARD PRIMER
•____ to the sequences amplified
• Hybridizes with the____ strand
5'
minus strand
27
• REVERSE PRIMER
•____ to the sequences amplified
• Hybridizes with the____ strand
3'
plus strand
28
PRIMER
• Binding to target is affected by: (2)
• _______of the forward and reverse primer must be similar
• Primer sequence (%GC)
• Length of strand
Melting temp (Tm)
29
DNA TEMPLATE
• From nucleotide extraction
• Routine clinical analyses requires:
______
100 ng - 1 ug
30
• Best templates (3)
• Good condition
• Free of contaminants
• No nicks and breaks
31
• Building blocks of DNA
• dNTPs - dATP (adenine), dTTP (thymine), dGTP (guanine), dCTP (cytosine)
DEOXYRIBONUCLEOTIDE BASES
32
DEOXYRIBONUCLEOTIDE BASES
• Usual requirement:
0.1-0.5 mM of each
33
DNA polymerase
• First polymerase from____
• Needed to be added after each round of_____
E. coli
denaturation
34
DNA polymerase
• Thermostable
• Good fidelity
• Taq polymerase
• Thermus aquaticus
35
DNA polymerase
• Also has reverse transcriptase activity
• Used in RT-PCR (RNA template)
• Tth polymerase
• Thermus thermophilus
36
• Provide optimal conditions for enzyme activity
• pH buffers and salts that provide cations
PCR BUFFER
37
• Provides pH for optimal enzyme activity and accurate amplification
• рН 8 - 9.5
• Can have accessory comoponents
Tris buffer
38
Tris buffer
• Provides pH for optimal enzyme activity and accurate amplification
• рН____
• Can have accessory comoponents
8 - 9.5
39
Binds inhibitors and stabilizes the enzyme.
Bovine serum albumin (10 to 100 m g/ mL)
40
Provides reducing conditions that may enhance enzyme activity.
Dithiothreitol (0.01 mM)
41
Lower the denaturing temperature of DNA with high secondary structure, thereby
increasing the availability for primer binding.
Formamide (1% to 10%)
42
May also reduce secondary structure to allow polymerase extension through difficult areas.
Triton X-100, glycerol, and dimethyl sulfoxide (1% to 10%)
43
Monovalent CATIONS
(KCl and (NH4)2SO4 )
44
(KCl and (NH4)2SO4 )
• Affect denaturing and
annealing temperatures
Monovalent CATIONS
45
• ↑ salt concentration = longer
DNA sequences denature
slower
MONOVALENT CATIONS
46
• Mg2+ is needed by DNA pol
• 1 NTP = 1 Mg atom
• If too low (↓ )
• By EDTA/other chelators
• ↓ amplicons
• If too high (↑)
• ↑ nonspecific products
divalent CATIONS
(MgCl2)
47
divalent CATIONS
• _____is needed by DNA pol
• 1 NTP = 1 Mg atom
• If too low (↓ )
• By EDTA/other chelators
• ↓ amplicons
• If too high (↑)
• ↑ nonspecific products
Mg2+
48
Machine:
• First PCRs
• Multiple water baths or heat blocks
• Done manually
thermal cycler
49
• Rapidly & automatically change to the required temperatures for each step and holds it there for designated periods
Thermal cyclers
50
CONTROL
Enzyme is active
Buffer is optimal
Primers are priming
Machine is working
Positive
51
CONTROLS
• Negative control without DNA
• "Contamination" control
Reagent Blank
52
CONTROLS
Negative control with DNA lacking the target sequence
Negative template
53
CONTROLS (3)
POSITIVE
REAGENT BLANK
NEGATIVE TRMPLATE
54
• Balance between aggressive amplification and avoidance of contaminating template
Contaminants
55
• Major cause of contamination:
• presence of….
• Can be resolved (2)
PCR products from previous amplifications
physically or chemically
56
PHYSICAL:
• Decontamination of pre-PCR areas
• Induces base damage that catalyzes the formation of single- and double-stranded breaks in DNA
• Enhanced effectivity with the addition of psoralens
UV Light
57
PHYSICAL:
• Enhanced effectivity with the addition of______
psoralens
58
• Widely used method for
decontamination and workspace prep
• Frequently wiped on surfaces that come in direct contact with specimen material to remove most DNA contamination
CHEMICAL: 10% Bleach
59
BLEACH SOLUTION
Chemical: dUTP-UNG system
60
• Substitutes the dTTP for dUTP in the PCR reagent master mix
• Uracil-N-glycosylase (UNG) degrades any nucleic acid containing uracil
• Incubation period at 50°C for 2-15 min added before PCR amplification
Chemical: dUTP-UNG system
61
Chemical: dUTP-UNG system
• Substitutes the dTTP for dUTP in the PCR reagent master mix
• Uracil-N-glycosylase (UNG) degrades any nucleic acid containing uracil
• Incubation period at___________ added before PCR amplification
50°C for 2-15 min
62
Concerning the primer
• Aberrant binding of the primer
•Carries the primer sequence and becomes a target for the next amplifications
MISPRIMES
63
Concerning the primer
• Artifact in PCR
• PCR products double the size of primers
• Primers binding to each other
PRIMER DIMERS
64
______has some activity at room temperature (22-25°C)
Primers can bind sequences other than their exact complements on the target (low stringency)
Taq pol
65
SOLUTION 1:______
• Good primer design
• Optimal amplification conditions
Solution 2:______
• Type 1: Reaction mixes are prepped on ice and placed in a prewarmed thermal cycler
• Type 2: Wax barrier
Wax separates the enzyme and templates from the primers
• Type 3: Sequestered enzymes
• Supplied in inactive form
Good Preparation
Hot-start PCR
66
Product Clean-up
AGAROSE GEL ELECTROPHORESIS
• Resolving amplification products
• Agarose digestion with_____ or ____
B-agarase or with iodine incubation
67
Product Clean-up
Residual component removal
• Using spin columns
•_____+______
Shrimp alkaline phosphatase
(SAP) + exonuclease I (Exol)
68
PCR Modifications
More than one primer pair added to PCR that are primed simultaneously
Multiplex PCR
69
Advantage: Many in one go!
Disadvantage: Inefficient & with cross-reactivity
Multiplex PCR
70
***Two pairs of primers*** used to amplity a single target in separate PCR programs
Nested PCR
71
Advantage: Increased sensitivity & specificity
Disadvantage: Time-consuming
Nested PCR
72
Starting material is RNA and converted to DNA by reverse transcriptase
Used in gene expression studies
"RT-PCR"
Reverse transcriptase por
73
Detects how much of the target sequence is present usually by fluorescence
Useful in detecting gene copy numbers, viral load, tumor load, and effects of treatment
"rt-PCR"
REAL-TIME PCR
74
• Replaced Ethidium bromide (EtBr)
• Highly specific and robust fluorescence
• Less toxic than EtBr
• Cheaper than Taq Man
SYBR Green I Assay
75
• Fluorescence occurs once the dye is separated from the quencher
• The probe makes it more specific than SYBR green
• Produces higher quality results
TaqMan Assay
76
Binds to the minus strand (template strand)
Located 5’ to the sequence to be amplified
Forward Primer
77
Binds to the plus strand (complementary strand)
Located 3’ to the sequence to be amplified
Reverse Primer
78
(dNTPs)
Deoxyribonucleotide Triphosphates
79
are the building blocks of DNA that are incorporated into the new DNA strand during extension.
dNTPs
80
Types of dNTPs in PCR:
• → Adenine (A)
• → Thymine (T)
• → Guanine (G)
• → Cytosine (C)
dATP (deoxyadenosine triphosphate)
dTTP (deoxythymidine triphosphate)
dGTP (deoxyguanosine triphosphate)
dCTP (deoxycytidine triphosphate)
81
is the enzyme responsible for synthesizing new DNA strands by adding dNTPs to the 3’ end of the primer.
DNA polymerase
82
First DNA Polymerase Used in PCR:
• Originally, ____was used.
• However, it was heat-sensitive, meaning it had to be added after each
E. coli DNA polymerase
83
• Most commonly used DNA polymerase in PCR.
• Thermostable: Can withstand high temperatures (94-96°C) during denaturation.
• Good fidelity (accuracy) but lacks proofreading ability (may introduce errors).
Taq Polymerase (Thermus aquaticus)
84
• Has reverse transcriptase activity, meaning it can synthesize DNA from an RNA template.
• Used in RT-PCR (Reverse Transcription PCR) to amplify RNA.
Tth Polymerase (Thermus thermophilus)
85
• Maintains a stable pH range of 8.0-9.5, which is ideal for DNA polymerase activity.
• Ensures that DNA strands remain stable during amplification.
• Prevents pH fluctuations that could inhibit the enzyme.
Tris Buffer
86
Monovalent Cations (KCl, (NH4)2SO4)
KCl (Potassium chloride)
(NH4)2SO4 (Ammonium sulfate)
87
Contamination control methods fall into two categories:
1. – Prevents contamination by separating areas, using UV light, and controlling workflow.
2. – Destroys contaminant DNA using bleach or enzymatic degradation methods.
Physical Decontamination
Chemical Decontamination
88
Separation of Pre-PCR and Post-PCR Areas
Physical Methods of Decontamination
89
occurs when primers bind to non-target regions of the DNA, leading to nonspecific amplification.
Mispriming
90
Causes of Mispriming
1. Low stringency conditions
2. Poor primer design
3. Contaminant DNA
